Vaccines effectively reduce and prevent death and disease from many viral infections such as for example flu, measles, mumps, smallpox, yellow fever.
Many viral vaccines are currently produced on embryonated chicken eggs or on primary chicken embryo fibroblasts isolated from chicken embryos. However, vaccine production occasionally has been complicated by inadvertent contamination with adventitious agents that may have originated from avian cell substrates used to propagate vaccine strains. Indeed reverse-transcriptase (RT) activity, an indication of the presence of retroviruses, was detected in chick cell-derived live, attenuated vaccines including those produced by European and US manufacturers for yellow fever, Measles and Mumps (Hussain et al., 2003, J. Virol 77:1105-1111; Johnson et Heneine, 2001, J. Virol., 75:3605-3612). Investigations of the origin of RT activity in those vaccines found evidence of particles containing RNA from endogenous avian leucosis virus (ALV-E) and endogenous avian virus (EAV) (Johnson et Heneine, 2001, J. Virol 75:3605-3612; Tsang et al., 1999, J. Virol 73:5843-5851; Weissmahr et al., 1997, J. Virol 71:3005-3012).
Both ALV-E and EAV are members of endogenous retrovirus families present in the chicken germ line. ALV-E are expressed from ev loci, which are inheritable proviral elements. Based on their envelope sequences, ALV-E are differentiated from ALV subgroups A to D and J which are exogenously acquired infections. While exogenous ALVs cause several neoplastic diseases, such as myocarditis and osteopetrosis in infected chickens, ALV-E are not known to be pathogenic to chickens. The lack of oncogenic potential with ALV-E infections may be attributed to the absence of both a viral oncogene and enhancer activity in the endogenous long terminal repeat (LTR). More than 20 different ev loci have been identified in White Leghorn chickens (ev-1 to ev-22). Ev loci designations are assigned in the order discovered and are phenotypically categorized with regard to the gene products they express and their capacity to generate infectious particles. ALV-E phenotypes conferred by ev loci range from structurally and enzymatically complete infectious particles to structurally or enzymatically (RT-) defective to no detectable viral protein expression. Most ev loci are structurally incomplete and therefore do not encode all sequences necessary for production of infectious virus particles. Chicken strain, named ev-0, has been obtained by breeding to be resistant to ALV-E. Line-0 chickens are lacking ev loci (i.e ev-0) but EAV proviral sequences are present in the genome line 0 chickens (Dunwiddie and Far as, 1985, Proc Natl. Acad. Sci. USA, 82: 5097-5101).
Little is known about the EAV family, which is distinct from but related to ALV family. EAV elements are present in at least 50 copies per chicken genome. However, none of the known EAV sequences represents full-length and intact retroviral genomes and no infectious EAV isolates have been yet identified. However EAV have been shown to be highly expressed in embryonic cells derived from the avian genus, gallus. Weissmahr et al. (1997, J. Virol 71:3005-3012) have shown that particles from the EAV endogenous retrovirus family are most likely responsible for a large portion of the particles-associated RT activity found in the supernatants of cultured chick embryo fibroblasts.
The risk of inadvertent transmission is particularly high for live attenuated virus vaccine since they cannot be subjected to an inactivation procedure and most of them are injected into human, thus by-passing non-specific immune protection mechanisms. Thus, to ensure safety of vaccines for animal and human use, the cell substrates for vaccine production have now to be tested for the presence of replication-competent retroviruses that could be passed to animal or human hosts during immunization (WHO technical reports Series, 1994).
On the other hands, embryonated chicken eggs and primary chicken embryo fibroblasts production systems are associated with several serious limitations, including:                a lengthy, cumbersome and resource-consuming manufacturing process that requires the procurement and quality control of large quantities of eggs or CEFs for each individual production campaign;        the need in many cases to use costly specific pathogen free (SPF) chicken embryos;        the risks of insufficient supply of eggs in cases of epidemic infections in donor chicken flocks;        the inflationist costs associated with the use of bovine sera originating from BSE-exempt countries;        the inability to use eggs for the propagation of viruses that are highly virulent and lethal to chickens.        
There is therefore an urgent need to improve on the current viral vaccines production technologies based on eggs or chicken-embryonic fibroblasts. The development of cell-culture platforms as an alternative to the eggs and CEF production systems for the manufacture of viral vaccines is likely the most rapid and promising solution to overcome current vaccines production bottlenecks and time constrains. Moreover, the use of cell lines for manufacture of viral vaccines, instead of egg or CEF platforms, would have the additional following advantages in connection with the safety of the vaccine: no antibiotic additives present in the vaccine formulation; no toxic preservatives (such as thiomersal) needed; reduced endotoxin levels, no egg allergy issue; no risk of adventitious agent/BSE by cell culture in protein and serum free media; higher purity of virus vaccine preparation.
Examples of cell lines for the production of viral vaccines are MDCK (cells derived from the kidney of Madin-Darby dog), PerC6 (cells derived from human embryonic retinal cells genetically modified by inserting the E1 genes from the human adenovirus type 5) developed by CRUCELL (Netherland)), VERO (cells derived from epithelial cells of kidney from African green monkey (Cercopithecus aethiops) isolate at the Chiba University in Chiba, Japan), BHK21 (Cells immortalized from baby hamster kidney cells). None of the cell lines available fulfil all the medical, regulatory and industrial requirements. For example, most of these cell lines are tumorigenic and there are important regulatory concern about the use of tumorigenic cells for the production of human vaccines; therefore, today the regulatory authorities are reluctant to approve tumorigenic cell substrates to produce mass vaccines. In addition, some of these cell lines are anchorage-dependant, which constitutes a serious hurdle for the industrial scaling-up of the vaccine production.
Therefore, there is a need to develop anchorage-independent cell lines, free of replication competent of retroviruses, that are non-tumorigenic and industrially compliant, which is susceptible to infection with a wide range of viruses. This is the purpose of the instant invention.
Thus, the inventor has taken advantage of its expertise in avian biology and in avian embryonic stem (ES) cells to undertake the development of novel stable duck cell lines that enables the efficient replication of a large group of human and veterinarian vaccines and vaccine candidates. By adapting a proprietary process (see WO 03/076601 and WO 05/007840), the inventor was able to generate a series of well characterized and documented duck cell lines (i.e the dEBx® cells) that are derived from duck ES cells, with no steps of genetic, chemical or viral immortalization and that do not produce replication-competent retroviruses in culture.